Prostate cancer is a leading cause of death in men, particularly in men over 60 years of age. The biological aggressiveness of the cancer varies greatly between individuals, with some cancers remaining as latent tumors which do not progress to clinical significance, and others rapidly progressing and metastasizing to a fatal disease within a few years. Clinical diagnosis and staging of prostate tumors has relied on digital rectal examination (DRE), computed tomography (CT) scans and/or endorectal magnetic resonance imaging (MRI). In addition, detection of cells bearing molecular markers specific to or highly expressed in prostate tissue has been used diagnostically.
Prostate specific antigen (PSA) is a protease made in high concentrations in prostatic epithelial cells and secreted into ducts of the prostate gland. PSA is a molecular marker for detection of prostate cancer (prostatic adenocarcinoma). Prostatic acid phosphatase (PAP) is another secreted enzyme that has been used as a serum marker for detection of prostatic metastases. A prostate-specific membrane antigen (PSMA), which is also highly expressed in prostate cancer tissues including bone and lymph node metastases, has been characterized, isolated and the gene encoding it has been cloned (U.S. Pat. No. 5,538,866 to Israeli et al.; O'Keefe D. S. et al, 1998, Biochim. Biophys. Acta 1443(1-2):113-127). Human glandular kallikrein-2 (hK2) is another prostate-associated protein that has been linked to prostate cancer (Partin A. W. et al., 1999, Urology 54(5): 839-845; Darson M. F. et al., 1999, Urology 53(5): 939-944).
Researchers have also associated the presence of prostate-specific markers with breast tissue and/or breast secretions (Yu H. & Berkel H., 1999, J. La. State Med. Soc. 151(4): 209-213). For example, detection of PSA in benign or malignant breast tumors and breast cyst fluids has been demonstrated (Diamandis, E. P. et al., 1994, Breast Cancer Res. Treat. 32: 291-300; Yu, H. et al., 1994, Clin. Biochem. 27: 75-79; Monne, M. et al., 1994, Cancer Res. 54: 6344-6347; Malatesta M. et al., 1999, Breast Cancer Res. Treat. 57(2): 157-163; Romppanen J. et al., 1999, Br. J. Cancer 79(9-10):1583-1587). Human kallikrein-2 is also expressed in breast tumors and normal breast secretions (Black M. H. et al., 2000, Br. J. Cancer 82(2): 361-367). Breast cancer affects about 10% of the U.S. female population and, therefore, detection of cancer markers associated with the disease has clinical utility.
Determining whether a cancer, such as prostate or breast cancer, is organ-confined, locally invasive (i.e., for prostate cancer, penetrating the capsule or seminal vesicle) or has metastasized to distant sites has significant impact on both the prognosis and determining the appropriate treatment of the cancer. Therefore, effective methods of detecting cancer metastasis are medically important. For example, detecting metastasis of prostate cancer to bone tissue or pelvic lymph nodes has been used in staging the progress of the disease. Metastatic prostate cancer cells at these sites may be detected by histological examination, PSA-specific immunocytology, or by reverse transcriptase-polymerase chain reaction (RT-PCR) to detect PSA mRNA (Deguchi, T. et al., 1993, Cancer Res. 53: 5350-5354). Prostate cancer cells are also presumed to be shed into the bloodstream, permitting them to disseminate to distant sites where the cells may become established as metastases. The presence of detectable prostate-specific antigen (PSA) and/or PSA-synthesizing cells in circulating blood is an abnormal situation indicative of potential prostate cancer metastases (sometimes referred to as “hematogenous micrometastasis”), although only about 0.01% of circulating solid tumor cells eventually result in a metastatic deposit (Moreno, J. G. et al., 1992, Cancer Res. 52: 6110-6112). Similarly, detecting abnormal amounts of PSA in females may indicate the presence of breast cancer (Yu H. & Berkel H., 1999, J. La. State Med. Soc. 151(4): 209-213).
Monoclonal antibodies that react with various prostate tissue antigens have been disclosed (U.S. Pat. No. 4,970,299 to Bazinet el al., U.S. Pat. No. 4,902,615 to Freeman et al., U.S. Pat. No. 4,446,122 and U.S. Re. Pat. No. 33,405 to Chu et al., U.S. Pat. No. 4,863,851 to McEwan et al., U.S. Pat. No. 5,055,404 to Ueda et al., U.S. Pat. No. 5,763,202 to Horoszewicz, and U.S. Pat. No. 5,773,292 to Bander). Monoclonal antibody-based immunoassays for measuring total PSA, free PSA (unbound to alpha-1-antichymotrypsin or “ACT”), and PSA-ACT complexes in body fluids have been disclosed for diagnostic methods to distinguish between patients with benign prostatic hyperplasia (BHP) and those with prostatic carcinoma (U.S. Pat. No. 5,614,372 to Lilja et al.; U.S. Pat. Nos. 5,698,402 and 5,710,007 to Luderer et al.). Other known immunoassays measure total serum PSA and distinguish between free PSA in serum and PSA-protein complexes which tend to be in higher concentrations in sera from prostate cancer patients (U.S. Pat. No. 5,672,480 to Dowell et al.) PSA concentrations in amniotic fluid, as determined by antibody-based assays, have also been correlated with gestational times as an indicator of fetal abnormalities (U.S. Pat. No. 5,579,534 to Diamandis).
RT-PCR detection of PSA-synthesizing cells in peripheral blood has also been correlated with stage D1 to D3 pathology, and with capsular penetration by prostate tumor cells (Moreno, J. G. et al., 1992, Cancer Res. 52: 6110-6112; Katz, A. E. et al., 1994, Urology 43: 765-775; U.S. Pat. Nos. 5,506,106, 5,688,649 and 5,674,682 to Croce et al.; Vessella, R. L. et al., 1992, Proc. Am. Soc. Cancer Res. 33: Abstract No. 2367; Diamandis, E. P. & Yu, H., 1995, Clin. Chem. 41:177-179). Generally, RT-PCR assays rely on obtaining RNA from a blood sample, reverse transcribing the RNA into cDNA, amplifying the cDNA using a pair of primers complementary to separate regions of the PSA gene, and demonstrating the presence of the amplified DNA by observing a particular size DNA on a gel. PCR amplification of DNA requires a repeated series of thermal denaturation, primer annealing and synthesis steps (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800159 to Mullis et al.). The amplified DNA may be further characterized by restriction endonuclease digestion, probing with a PSA-specific oligonucleotide (e.g., Southern blotting), and/or DNA sequencing. Micrometastasis of other types of solid tumors (melanoma, neuroblastoma, breast cancer, cervical cancer) has also been detected using RT-PCR assays for other cell markers (Wu, A. et al., 1990, US & Can. Acad. Pathol. Ann. Mtg., Abstract No. 641.;Smith, B. et al., 1991, Lancet 338: 1227-1229; Naito, H. et al., 1991, Eur. J. Cancer 27: 762-765; Mattano, Jr., L. A. et al. 1992, Cancer Res. 52: 4701-4705; U.S. Pat. Nos. 5,543,296 and 5,766,888 to Sobel et al.).
PSA is a member of a group of serine proteases known as glandular kallikreins. The human kallikreins include pancreatic/renal kallikrein (hK1), prostate-specific glandular kallikrein (hK2 or HPSK), and PSA (also known as hK3) which are encoded by related genes (hKLK1, hKLK2 and hKLK3 or PSA, respectively). Because of the chemical and structural similarities of these proteins and genes, it is important to be able to distinguish the individual proteins in immunoassays and the individual genes or their corresponding mRNA in nucleic acid detection methods. Expression of hK2 has been associated with prostate and breast cancers (Black M. H. et al., 2000, Br. J. Cancer 82(2):361-367; Partin A. W. et al., 1999, Urology 54(5): 839-845; Darson M. F. et al., 1999, Urology 53(5): 939-944). Antibodies that react specifically with human prostate-specific glandular kallikrein (anti-hK2) but not with PSA have been described (U.S. Pat. No. 5,516,639 to Tindall et al.; U.S. Pat. No. 5,786,148 to Bandman et al.). The gene sequences for human kallikreins are known (U.S. Pat. No. 5,786,148 to Bandman et al.; GenBank Accession Nos. NM005551, M21895, M27274, M24543, M21897, M26663, M21896, S75755, U17040, S39329 and M18157).
Some nucleic acid sequences useful for amplification of PSA mRNA in RT-PCR assays and detection of DNA products have been described (e.g., in Deguchi, T. et al., 1993, Cancer Res. 53:5350-5354; Katz, A. E. et al., 1994, Urology 43: 765-775; Moreno, J. G. et al., 1992, Cancer Res. 52: 6110-6112; U.S. Pat. Nos. 5,506,106, 5,688,649 and 5,674,682 to Croce et al.). Detection of prostate-associated genetic markers (e.g., PSA, PSMA and/or hK2) at locations outside of prostate tissue is useful for detecting cancer metastases, particularly prostate cancer in men and breast cancer in men and women, thereby indicating appropriate treatment. Thus, there exists a clinical need for nucleic acid sequences and methods that are used to specifically detect the presence of genetic expression of prostate-associated genetic markers, i.e., specific mRNA sequences that provide diagnostic information. There is a particular need for detecting these prostate-associated marker mRNA at levels useful for detecting relatively few cells containing the mRNA in a biological sample, such as occur in micrometastases.